Lower motoneuron death is believed to be the primary defect in spinal muscular atrophy (SMA), a childhood hereditary neuromuscular disease almost as prevalent as cystic fibrosis, perhaps the best-known genetic disease in children. Muscle denervation and atrophy ensue as a result of motoneuron loss. There is no current cure for SMA. Mutations in the survival of motoneuron 1 (SMN1) gene account for SMA. All cells in the body produce SMN and its major function is in spliceosome assembly. Clinically, SMA exhibits several degrees of severity from lethal to mild. This is explained by the presence of a second SMN gene in the human genome (SMN2). SMN2 is essentially identical to SMN1 except for a mutation that causes exon skipping in the splicing of 90% SMN2 RNA, leading to the production of an unstable, minimally functional protein (SMN 7). Only 10% of SMN2 transcripts code for a functional SMN protein. SMN2 can exist in multiple copies, hence the more SMN2 copies the less severe SMA. Although mice only harbor one SMN gene, they have been used to model human SMA by introducing multiple copies of human SMN2. Mouse homozygous for a deletion in their SMN gene (Smn-/-) are embryonic lethal. Smn-/- mice with two copies of SMN2 die around 4-5 days after birth and show features of the most severe human disease (i.e. type I SMA). Addition of a cDNA for SMN?7 to the genotype of type I mice, improves survival to 14 days in average (type II mice). Novel type II mice, in which the targeted Smn allele can be reverted to a functional one following Cre-recombination have been generated. They show similar survival and phenotype as standard type II mice. Type I mice were genetically rescued by crossing them with transgenic mice expressing normal SMN driven by a pan-neuronal promoter. Muscle fiber-specific expression of normal SMN was insufficient to rescue type I mice. Thus, these results indicate that neurons are the targets of SMN deficiency in type I SMA mice but they do not distinguish whether, like in the human disease, the deficiency occurs primarily in motoneurons. If so, restoration of normal SMN levels selectively in motoneurons should have great positive impact on the survival and phenotype of the model SMA mice. Here, we will test this prediction. In Aim 1, we will use transgenic mice that we have generated, in which human SMN expression is driven by the motoneuron-selective Hb9 promoter, to test whether their crossing into type I mice can extend their survival and rescue their SMA-like phenotype. In Aim 2, we will use a complementary approach that will attempt rescuing the novel type II mice by crossing them to Hb9-Cre animals, so that endogenous SMN expression will be restored selectively in motoneurons following Cre inversion of the special Smn targeted allele in these animals. Results from the experiments proposed here will: (i) clarify the role of motoneurons in SMA mouse models, (ii) validate therapeutic approaches that use purified motoneuron cultures in high throughput screening for molecules that increase SMN levels in humans. PUBLIC HEALTH RELEVANCE: Human SMA results from motoneuron loss that is accompanied by muscle atrophy, caused by low levels of SMN protein. Although mouse models of SMA recapitulate many features of the human disease, it is still unclear whether their phenotypes are primarily due to motoneuron deficits. Results from the experiments proposed will clarify the role of motoneurons in SMA mouse models, and validate therapeutic approaches that use purified motoneuron cultures in screening for agents that increase SMN levels in humans.